Phase I trial of intramuscular injection of a recombinant adeno-associated virus serotype 2 alphal-antitrypsin (AAT) vector in AAT-deficient adults.

A phase I trial of intramuscular injection of a recombinant adeno-associated virus serotype 2 (rAAV2) alpha1-antitrypsin (AAT) vector was performed in 12 AAT-deficient adults, 10 of whom were male. All subjects were either homozygous for the most common AAT mutation (a missense mutation designated PI*Z) or compound heterozygous for PI*Z and another mutation known to cause disease. There were four dose cohorts, ranging from 2.1 x 10(12) vector genomes (VG) to 6.9 x 10(13) VG, with three subjects per cohort. Subjects were injected sequentially in a dose-escalating fashion with a minimum of 14 days between patients. Subjects who had been receiving AAT protein replacement discontinued that therapy 28 days before vector administration. There were no vector-related serious adverse events in any of the 12 participants. Vector DNA sequences were detected in the blood between 1 and 3 days after injection in nearly all patients receiving doses of 6.9 x 10(12) VG or higher. Anti-AAV2 capsid antibodies were present and rose after vector injection, but no other immune responses were detected. One subject who had not been receiving protein replacement exhibited low-level expression of wild-type M-AAT in the serum (82 nM), which was detectable 30 days after receiving an injection of 2.1 x 10(13) VG. Unfortunately, residual but declining M-AAT levels from the washout of the protein replacement elevated background levels sufficiently to obscure any possible vector expression in that range in most of the other individuals in the higher dose cohorts.

Use of recombinant vaccinia to restimulate antigen specific human peripheral blood cytotoxic T lymphocytes.

A simple method is described for using recombinant vaccinia virus for restimulating human peripheral blood mononuclear cells (PBMC) to generate antigen specific CD8 + alpha beta cytotoxic T cell (CTL) effector populations. Effector PBMC were restimulated in vitro with a subpopulation of PBMC, which had been infected with recombinant vaccinia at very low multiplicities of infection in the absence of serum. This protocol avoided the vaccinia mediated overt cytopathic effects on the effector PBMC and generated bulk CTL cultures, which could be used for the identification of epitopes recognised by antigen specific CTL.

Performance of three generations of anti-hepatitis C virus enzyme-linked immunosorbent assays in donors and patients.

BACKGROUND: Prevention of posttransfusion non-A,non-B hepatitis in recipients of blood components improved considerably with the introduction of the second-generation of hepatitis C virus (HCV) antibody tests. In 1993, third-generation HCV antibody assays were introduced in Europe. STUDY DESIGN AND METHODS: The performance of three generations of anti-HCV enzyme-linked immunosorbent assay (ELISA) (ELISA-1, -2, -3) was compared in routine blood donor screening (99,394 donations were tested with ELISA-1, 167,999 donations with ELISA-2, and 262,090 donations with ELISA-3) and in serial samples from nine patients with documented acute posttransfusion HCV infection. RESULTS: Eight (0.01%) repeat donors, previously negative in ELISA-1, were found positive in ELISA-2 and were confirmed as positive in second-generation recombinant immunoblot assay and/or cDNA polymerase chain reaction. In the donor population, no difference in the sensitivity of ELISA-2 and -3 was observed. The specificity of the three generations of ELISAs was comparable (99.8, 99.7, and 99.7%). In seroconversion samples, ELISA-2 and -3 detected HCV antibodies at the same time in seven patients, but in two patients, ELISA-3 found HCV antibodies, respectively, 63 and 77 days earlier than ELISA-2 did. In the seroconversion samples, ELISA-2 and -3 were significantly more sensitive than second- and third-generation recombinant immunoblot assays. CONCLUSION: ELISA-3 did not detect more HCV-infected individuals in a donor population that previously tested negative in ELISA-2, but it did detect HCV antibodies earlier in some patients with acute HCV infection. ELISA-2 and -3 were significantly more sensitive than second- and third-generation recombinant immunoblot assays.

Germline transmission of exogenous genes in chickens using helper-free ecotropic avian leukosis virus-based vectors.

We have used vectors derived from avian leukosis viruses to transduce exogenous genes into early somatic stem cells of chicken embryos. The ecotropic helper cell line, Isolde, was used to generate stocks of NL-B vector carrying the Neo(r) selectable marker and the Escherichia coli lacZ gene. Microinjection of the NL-B vector directly beneath unincubated chicken embryo blastoderms resulted in infection of germline stem cells. One of the 16 male birds hatched (6.25%) from the injected embryos contained vector DNA sequences in its semen. Vector sequences were transmitted to G1 progeny at a frequency of 2.7%. Neo(r) and lacZ genes were transcribed in vitro in chicken embryo fibroblast cultures from transgenic embryos of the G2 progeny.

The role of organ vascularization and lipoplex-serum initial contact in intravenous murine lipofection.

Following intravenous administration of cationic lipid-DNA complexes (lipoplexes) into mice, transfection (lipofection) occurs predominantly in the lungs. This was attributed to high entrapment of lipoplexes in the extended lung vascular tree. To determine whether lipofection in other organs could be enhanced by increasing the degree of vascularization, we used a transgenic mouse model with tissue-specific angiogenesis in liver. Tail vein injection of N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP)/cholesterol lipoplexes resulted in increased lipoplex entrapment in hypervascularized liver but did not boost luciferase expression, suggesting that lipoplex delivery is not a sufficient condition for efficient organ lipofection. Because the intravenously injected lipoplexes migrated within seconds to lungs, we checked whether the effects of immediate contact with serum correlate with lung lipofection efficiency of different DOTAP-based formulations. Under conditions mimicking the injection environment, the lipoplex-serum interaction was strongly dependent on helper lipid and ionic strength: lipoplexes prepared in 150 mM NaCl or lipoplexes with high (>33 mol%) cholesterol were found to aggregate immediately. This aggregation process was irreversible and was inversely correlated with the percentage of lung cells that took up lipoplexes and with the efficiency of lipofection. No other structural changes in serum were observed for cholesterol-based lipoplexes. Dioleoyl phosphatidylethanolamine-based lipoplexes were found to give low expression, apparently because of an immediate loss of integrity in serum, without lipid-DNA dissociation. Our study suggests that efficient in vivo lipofection is the result of cross-talk between lipoplex composition, interaction with serum, hemodynamics, and target tissue "susceptibility" to transfection.

Development of a competitive PCR method for in vitro and in vivo quantification of herpes simplex virus thymidine kinase and neomycin resistance-expressing cells used in a clinical trial.

The aim of this study was to set up a sensitive and specific method to quantify the number of gene-modified cells in a gene therapy clinical trial currently underway at our institution. This trial involves the use of retrovirally transduced allogeneic T cells expressing the herpes simplex-1 thymidine kinase (HSV-TK) and neomycin-phosphotransferase (NeoR) resistance gene. Quantification by competitive PCR was performed, with two homologous internal standards (deltaTK, deltaNeoR), 30 bp shorter than the target sequences (TK, NeoR), coupled to fluorescent laser-based detection. Assessment of the amplification systems procedures was carried out for each sequence. The 30-bp deletion did not affect the amplification efficiency significantly. Determination of the plateau phase of both amplified sequences demonstrated that each sample must be quantified during the predetermined exponential phase. Finally, a blinded study of a transduced cell dilutions panel validated the overall methodology. The competitive PCR was applied to quantification of the retroviral transduction process by quantifying the NeoR gene in transduced PBMC samples (prior to G418 selection) from 18 donors in our clinical trial. A mean transduction efficiency of 9.78% +/- 1.37% was observed. We also quantified TK-expressing donor transgenic T cells in a murine GvHD model. Results demonstrated on initial expansion of donor HSV-TK- expression T cells as well as a significant ganciclovir (GCV)-induced decrease correlated with the number of circulating gene-modified T cells. Therefore, we have developed an efficient gene quantification tool that should be useful for in vivo monitoring of gene-modified cells.

Systemic circulation of poly(L-lysine)/DNA vectors is influenced by polycation molecular weight and type of DNA: differential circulation in mice and rats and the implications for human gene therapy.

Effective gene therapy for diseases of the circulation requires vectors capable of systemic delivery. The molecular weight of poly(L-lysine) (pLL) has a significant effect on the circulation of pLL/DNA complexes in mice, with pLL(211)/DNA complexes displaying up to 20 times greater levels in the blood after 30 minutes compared with pLL(20)/DNA. It is shown that pLL(20)/DNA complexes fix mouse complement C3 in vitro, independent of immunoglobulin binding; are less soluble in the blood in vivo; bind erythrocytes; are rapidly removed by the liver, where they associate predominantly with Kupffer cells; and result in a rapid increase in hepatic leukocytes expressing high levels of complement receptor 3 (CR3). The circulation properties of these complexes are also dependent on the type of DNA used, with circular plasmid DNA complexes exhibiting increased circulation compared with linear DNA. PLL(211)/DNA complexes bind erythrocytes and associate with Kupffer cells but, in contrast, do not fix mouse complement in vitro and are unaffected by the type of DNA used. In rats, both types of complexes produce hematuria and are rapidly removed from the circulation. Correlation of in vivo and in vitro results suggests that the solubility of complexes in physiological saline and species-matched complement fixation and erythrocyte lysis may correlate with systemic circulation. Analysis using human blood in vitro shows no hemolysis, but both types of complexes fix complement and bind IgG, suggesting that pLL/DNA complexes may be rapidly cleared from the human circulation.